The fourth component of human complement is encoded by two separate but closely-linked loci within the major histocompatibility complex on the sixth chromosome. The primary goal of the proposed studies is to determine the structural differences between C4 molecules produced by the two loci and to determine the relationship of these structural differences to the differences in specific functional activity of the C4 produced by the two loci. C4 will be isolated from individuals who express C4 variants on each of the loci which code for C4. The C4 isolated from different individuals will then be compared and the peptide or peptides containing the probable amino acid substitutions responsible for the genetic difference will be isolated and sequenced. Current data suggest that the genetic differences in C4 are all contained with the alpha chain, the largest of the three polypeptide chains which make up the C4 molecule. Intact isolated C4 will be cleaved with Cls to release the C4a peptide. The remaining alpha1- polypeptide chaind will be cleaved with the C3b inactivator in the presence of C4 binding protein. This results in three alpha-derived peptides (molecular weights 45,000, 28,000, 18,000) which will be isolated, and their amino terminal sequences will be determined. They will then be subjected to analytical peptide maps by high performance liquid chromatography after digestion with trypsin, chymotrypsin and elastase. The peptides responsible for the antigenic (Chido and Rodgers antigens) and charge differences on C4 encoded by the two loci will then be isolated and their amino acid sequences determined. The specific activity and stability of C4 encoded by the two loci will be compared through the use of hemolytic asays and direct demonstration of cleavage of C4 during actvation and inactivation. The proposed studies will aid in understanding the role of C4 in normal complement activation and its possible relationship with other gene products of the majhor histocompatibility complex.